1. Field of the Invention
The general field of this invention is that of free-fluid electrophoresis. More specifically, the invention concerns the combination of the high separation efficiency of capillary zone electrophoresis (CZE) with the high operational efficiency similar to high performance liquid chromatography (HPLC) to create a new computer-controlled separation process, namely chromatography-format fluid electrophoresis (CFFE).
2. Description of Related Art
The primary technologies used for high-resolution separation of biological proteins and macromolecules have been variations of either chromatography or electrophoresis. In chromatography, separation occurs because of selective interactions between sample molecules and the fluid or solid phases through which the fluid sample is transported by either gravity or pressure. In electrophoresis an electrical field is used to impart motion to electrically charged sample molecules which then separate according to their mobility.
Early in the 20th century a Russian botanist, Mikhail Tswett, first developed chromatographic techniques to separate plant pigments. Chromatography has since evolved into techniques as diverse as one- and two-dimensional thin layer chromatography and various forms of liquid chromatography (LC), e.g., adsorption, affinity, ion exchange, gel permeation, and size exclusion. This wide variety of available techniques reflects the lack of a single instrument or method that satisfies the needs of the entire scientific community. When the research scientist wants a sample purified and/or analyzed, the method of choice is established based upon either past experience with similar samples or the result of trial and error with the wide variety of devices in the laboratory.
A number of chromatography variants have evolved because of the wide diversity of sample molecules and compatible liquid environments. For example, ion exchange chromatography is capable of separating molecules differing by very small differences in charge, depending upon ionic interactions with the media through which they pass. Affinity chromatography is a special type of adsorption chromatography in which the molecule to be analyzed or purified is specifically and reversibly adsorbed to a complimentary binding site on an insoluble porous solid substructure, the matrix. A change in solvent conditions may be used later to release the bound molecules.
Speed of chromatographic separation is second in importance only to the resolution of the separation; increased speed can be achieved by driving the solute through the matrix at a higher velocity than normal gravity-induced sedimentation. Generally the solute is driven by applying a high pressure, hence the name high pressure LC, an early name for the method now called HPLC which has evolved into the method of choice for high-resolution fractionation of biological materials. The development and optimization of HPLC was led by chemists who appreciated the overall versatility and flexibility of chromatography and who were experienced with both LC and thin layer chromatography. Although these scientists also had access to gel electrophoresis, HPLC identified and solved the major difficulties surrounding requirements for increased quantities of purified products.
Although, currently, electrophoresis is absolutely critical for certain analysis such as that involved in gene sequencing, it is generally much less popular than chromatography, especially for preparative separations. A major drawback to electrophoresis has been ohmic heating caused by the current flow and the resulting thermal degradation or disruption of the separation process. Early attempts at fluid electrophoresis date back to Reuss in 1807 and were greatly hampered by thermal convection. Modern electrophoresis was developed as an analytical and preparative technique by Arne Tiselius in 1937.
However, electrophoresis did not become practical and popular until a matrix was introduced to limit convective mixing. First, paper-based electrophoresis was used in the 1950's to elucidate the biochemical pathways of carbon fixation in green plants. In the 1960's electrophoresis in a gel matrix was introduced in starch, agarose, and polyacrylamide forms and quickly revolutionized protein biochemistry. In gel electrophoresis the electric field simply serves to move the sample through the gel matrix while the sieving effect of the matrix eliminates convection and improves the separation. Therefore, strictly speaking, gel electrophoresis is a hybrid between electrophoresis and chromatography with the matrix being responsible for at least part of the resolution of the separation. Gels with highly uniform pore structure provide size exclusion (i.e., separation based on molecular size) as well as limiting flows due to thermal convection. Isoelectric focusing, which takes advantage of the molecular mobility dependence on pH, combined with "ordinary" electrophoresis as a two-dimensional analog of thin layer chromatography has been an important analytical diagnostic technique.
Historically most separation processes, both chromatographic and electrophoretic, have been carried out in a supporting matrix which also served as the discriminator producing the separation. It must be kept in mind that matrix-based electrophoretic techniques generally combined chromatographic separation with true electrophoresis. However, the disruptive effects of thermal convection in free-fluid electrophoresis have been exaggerated. Although Hjerten's.sup.(11) classic 1967 work is often cited as an indictment of thermal convection, Hjerten employed rotation of his separation tube to control sedimentation, not thermal convection. HPLC, a largely free-fluid separation, has side-stepped the problem of thermal convection for the most part, since there is very little heating associated with fluid flow. On the other hand, electrochromatography is similar to gel electrophoresis in that considerable ohmic heating is developed but the packing material (matrix) is sufficiently restrictive to preclude convective mixing.sup.(12).
Like chromatography electrophoresis has also been used on a preparative scale. Preparative electrophoresis began when electrophoresis on filter paper was operated vertically and the dripping separands were collected over time. Exchanging a thin flowing liquid curtain for the paper removed the size exclusion advantage of the paper but improved the overall unrestricted separation of the separand molecules. "Continuous flow" electrophoresis grew from initial systems in 1960 until newer methods, such as HPLC, were shown to be easier to operate. With the perfection of HPLC, continuous flow electrophoresis declined from being the primary system for purification and collection of proteins and cells to a minor system used only in highly specialized cases, relying on older equipment that is no longer even manufactured.
During the 1970's, a number of electrophoresis systems with vertical cylindrical columns were developed to accomplish preparative batch separations. The separations took place in a thin annular part of the column since the central core and outer surface were cooled to minimize heat build-up. Density gradients of various high molecular weight gel polymers were frequently added to stabilize the migrating bands against thermal convection.
Although electrophoresis is conceptually simpler, chromatography has generally won the popularity contest. The reigning monarch of chromatography is HPLC which is capable of both sensitive analytic separations as well as preparative scale separations. No single analog to HPLC has thus far evolved in electrophoretic separations, although specific apparatus is available for some applications. CZE, the latest electrophoretic method to challenge HPLC, solves the problem of thermal mixing by making the separation column diameter so small that the wall effectively dampens appreciable thermal mixing. Also, the column cross section is so small that thermal gradients are kept very small.
In CZE the buffer and separation molecules are transported through the capillary by a process known as electroosmosis. When an electric field is applied to the filled capillary, the fluid will move relative to the charged inner surface. The cause of the charged inner wall of the capillary is ionization, ion adsorption, and/or ion dissolution due to contact with a polar medium. This wall condition can be described by a zeta potential. This inner surface charge is nominally negative in the presence of common aqueous buffers. Pharmaceuticals and proteins are frequently positively charged because of their pervasive amino functionality. The hydrated positive ions (counter-ions) at the wall move under the action of the electric field and through viscosity, causing a plug-type flow in the separation chamber (capillary). Unfortunately, positive sample species are also attracted to the negatively charged walls which can cause nonuniform wall zeta potential and hence an unpredictable electroosmotic (EOF) or "wall mobility."
CZE has now found a small niche due to its high resolving power and automated operation. Extremely small samples can be precisely separated by CZE in narrow-bore glass tubing--a significant advantage to those with only small, expensive, or scarce samples, CZE systems have found a market for drug testing and forensic applications and the "high end" equipment is now characterized as "high performance capillary electrophoresis."
In the analytical range CZE is considered superior, but attempts to provide CZE with preparative abilities, i.e., the capability of collecting useful quantities of separated samples.sup.(20) has yielded, at best, a tedious collection scheme not suited to commercial application. A major advantage of chromatography continues to be flexibility and versatility provided by a wide range of different columns and internal packings that can be adapted to a single front end (i.e., sampler/buffer insertion apparatus) and back end (i.e., detection, and fraction collection devices). However, this improved flexibility does come at a rather high system price.
The major disadvantage of chromatography is the necessity to obtain or find optimum adsorption materials to get the best resolution for a given separation. The power of affinity chromatography relies on highly specific interactions between the porous column matrix and the sample molecules. Ion exchange chromatography uses an insoluble matrix to which various charge groups are attached. The actual separation requires very selective sample attachment (adsorption) and detachment (desorption) from the matrix as different solutions are passed through the column. Thus, many chromatographic separations can be optimized only when molecular-specific interactions have been identified, matrix material manufactured, tested, and protocol for elution has been confirmed. Therefore, efforts to optimize the system can become very significant research tasks in themselves.
An additional problem with chromatographic separations of biological material is loss of biological activity. Close contact and physical/chemical interaction of the sample with the stationary phase provides a constant danger of less of biological activity (i.e., denaturation) as well as the significant possibility of irreversible adsorption of the sample to the chromatographic medium. Also, the use of the supporting matrix in HPLC leads to problems including "eddy migration" and adsorption interactions affecting resolution and separation time. For high resolution, HPLC suffers inherently from external (to the column) processes such as detection and injection; i.e., after injection the sample zone is subjected to hydrodynamic dispersion in the connecting tubing, and similar dispersion occurs when the separated zones leave the column on their way to the detector. In preparative HPLC the very high pressures necessary to ensure adequate throughput cause the stationary phase to deform or "slump." Also, due to the large pressure drop incurred by the column, the height-to-diameter ratio is small, often leading to unequal migration paths for the sample moving through the column.
Electrophoresis in thin layer gels is the world-wide standard for hospitals and diagnostic laboratories because of ease of operation and low cost of equipment. Medical technicians in hospitals and clinics use thin layer gel electrophoresis for the routine, standard analysis of body fluids. When properly stained and stored, the gels identify abnormal molecules and their relative quantity. A problem with gels is their lack of resolving power since gels can only be used to identify those proteins which bind the stain. Also, modern gel electrophoresis is an accumulation, for the most part, of manually intensive methodologies that cannot be run unattended and that cannot be readily automated: casting gels, applying samples, running gels, and staining gels are time-consuming tasks prone to irreproducibility and poor quantitative accuracy.
To electrophoretically distinguish the proteins and macromolecules not readily detected on gels, one must use CZE. An average commercial CZE system costs more than 20 times a typical gel electrophoresis system but will identify more than 20 times the number of subfractions under ideal conditions. Capillary systems now incorporate a variety of coatings and packing materials in their narrow bore (&lt;100 .mu.m) columns and separations of some mixtures of pharmaceutical interest have been resolved to the point that all molecules of interest have been identified. The small sample sizes (nanograms) can be an advantage if cost and availability limit the sample but are a disadvantage if further analysis of separated subfractions is necessary. The collection of subfractions is neither feasible nor practical with the presently available commercial units.
Reproducibility is an important factor in separations, especially analytical. In some cases it may be more important than resolution. Since the capillaries used in CZE are usually &lt;100 .mu.m in diameter, significant adsorption of the sample frequently takes place. This adsorption not only affects reproducibility but also resolution. As sample material coats the capillary walls, a nonuniform wall zeta potential results. This phenomenon has been investigated.sup.(18) where it was shown that plug flow in a capillary cannot be expected in cases where nonuniform wall mobilities exist, and further, that a nonuniform zeta potential generally leads to significant dispersion of subfraction peaks. It was shown that the nonuniform wall mobilities induced both sample circulations and parabolic flows in the affected capillaries. These sample circulations were highly localized and were not considered as significant a cause of peak broadening as were parabolic flows. Dispersion effects in the free-fluid electrophoretic process has led to a necessary but rather onerous preoccupation with capillary wall conditions. Much money, time, and effort have been spent in the development of wall coatings which reduce adsorption and enhance reproducibility of results.
Many problems are also associated with the methods of sample injection used in CZE. In a 1989 article, Grushka and McCormick.sup.(10) point out these inadequacies. These authors found that the actual insertion of the capillary into or withdrawal of the capillary from the sample solution resulted in the extraneous injection of sample into the capillary. They also found that the injected core length can exceed the maximum value permitted for the realization of the high separation efficiency of CZE. They concluded that the extraneous injection length will be superimposed on both the electrokinetic and hydrostatic injection techniques resulting in total injection zone sizes which can be unacceptable in terms of plate heights. Thus, while "dunking" the capillary might be simple, it is also quite crude with respect to obtaining the injected zone most desirable for high resolution.
In summary, chromatography and electrophoresis have distinct but different advantages for the separation and purification of biological materials. Scientists have traditionally purchased, used, and discarded a variety of instruments claimed to improve the purity of their desired product. Some of these instruments have been used successively to provide a slight improvement at each stage. It is the objective of this patent to take advantage of the best features of each method combined with specific innovations and produce a single instrument which will satisfy a majority of users' needs.